Endocannabinoid System and TRPV1 Receptors in the Dorsal Hippocampus of the Rats Modulate Anxiety-like Behaviors.

Objective(s) Fatty acid is amide hydrolase which reduce endogenous anandamide. Transient receptor potential vanilloid-1 (TRPV1) channels have been reported to have a role in the modulation of anxiety-like behaviors in rodents. In the present study, the effects of either endocannabinoid system or TRPV1 channels and their possible interaction on anxiety-like behaviors of the rats were explored. Materials and Methods Elevated plus-maze test of anxiety was used to induce anxiety. Capsaicin and AMG 9810 as TRPV1 agonist and antagonist respectively were injected into the dorsal hippocampus. URB 597 as selective FAAH inhibitor and AM 251 as CB1 receptor selective antagonist were also injected into the dorsal hippocampus. The effect of AMG 9810 on the response of URB 597 was also examined. Results Intra-CA1 injection of URB 597 (0.001, 0.01 and 0.1 µg/rat) and AMG 9810 (0.003, 0.03 and 0.3 µg/rat) produced anxiolytic-like effects. Intra-CA1 infusion of capsaicin (0.003, 0.03 and 0.3 µg/rat) increased the anxiety-related behaviors and AM 251 (0.001, 0.01 and 0.1 µg/rat) did not significantly change the animals behavior. AMG 9810 at the dose of 0.003 µg/rat did not change the anxiolytic-like effect of URB 597. Conclusion The results of the present study demonstrated that both endocannabinoid system and TRPV1 receptors may affect anxiety-like behaviors. In addition, it seems that TRPV1 receptors are not involved in the effects of anandamide on anxiety-related behaviors in the CA1 region.


Introduction
The endocannabinoid system has a modulatory role in several physiological processes, particularly in the brain (1,2). Studies suggest that by activating cannabinoid CB1 receptors, endocannabinoids play an important role in the control of anxiety (3). Anandamide is the best recognized of the endocannabinoids isolated so far. It is hydrolyzed within cells by fatty acid amide hydrolase (FAAH) (4). Therefore, selective inhibitors of FAAH enzyme such as URB 597 can magnify and prolong the duration of action of endogenously released anandamide. The anxiolytic-like effects of URB 597 in the isolation-induced ultrasonic vocalizations in rat pups and in adult rats tested in the elevated zero maze have been revealed (5).
Transient receptor potential vanilloid-1 (TRPV1) channel is a receptor that is activated by capsaicin, a pungent component of hot chili peppers, as well as by low pH and noxious heat (6). These receptors are involved in pain perception, regulation of body temperature, bladder function, itching and pulmonary diseases [for review see Moran et al (7)]. Furthermore, TRPV1 receptors are expressed in brain regions associated with control of emotional and stress responses including the hippocampus, medial prefrontal cortex, locus coeruleus and dlPAG (dorsolateral periaqueductal grey) (8,9). However, the role of TRPV1 receptors in the modulation of anxiety-like behaviors has not been explored well so far. In 2004, Kasckow et al (10) provided the first evidences on the anxiogenic-like effect of olvanil (TRPV1 receptor agonist) and the anxiolytic-like effect of capsazepine (selective TRPV1 receptor antagonist). These findings have also been repeated in some recent studies.
Until now, the selective and exclusive endogenous ligand (s) for TRPV1 receptors has not been discovered. However, it is now apparent that anandamide is also an endogenous ligand for these receptors (11). Although, CB1 and TRPV1 receptors are expressed in several brain regions but they may have opposite roles in the regulation of neural activity (11). CB1 is a receptor which inhibits adenylate cyclase and reduces neurotransmitter release in the central nervous system (12) but TRPV1 receptor promotes depolarization of the neurons (13). Thus, anandamide may interact with CB1 and TRPV1 receptors to inhibit or promote anxietylike behaviors, respectively. The functional consequences of the interaction between these receptors have rarely been explored (11). Hippocampus is a brain structure with an important role in the modulation of anxietyrelated behaviors and a high density of CB1 and TRPV1 receptors and FAAH enzyme exist in this area (8,14).
On the basis of the above evidences, the present study was designed to investigate the effects of a FAAH inhibitor and selective TRPV1 agonist and antagonist on anxiety-like behaviors in the CA1 region of the hippocampus and to investigate a possible interaction between endocannabinoids and TRPV1 receptors.

Animals
Male Wistar rats, weighing 200-250 g at the time of surgery, were used in the present study. Animals were housed 5 per cage, in a room with a 12:12 hr light/dark cycle (lights on 07:00 hr) and controlled temperature (23±2 o C). Animals had access to food and water ad libitum. All rats were handled three days prior to the behavioral testing for 5 min. Each experimental group included seven animals. All experimental procedures were carried out according to the local Animal Ethics Committee protocols.

Surgery
Rats were anesthetized intraperitoneally with ketamine hydrochloride (75 mg/kg) and xylazine (4 mg/kg) and fixed in a stereotaxic frame. The stainless steel guide cannula (23gauge) was implanted unilaterally in the right dorsal region of the hippocampus (CA1, coordinates: AP: -3.30 mm; L: 1.8 mm; D: -3 mm) according to Paxinos and Watson (15). It was then fixed to the skull with acrylic dental cement.

Procedure
Intra-CA1 injections were made using a microinfusion pump (KD Scientific, Holliston, MA, USA). Injector (30-gauge) was glued to polyethylene tubing filled with distilled water. The tubing was connected to a 10-μl microsyringe mounted on the microinfusion pump. Rats were hand-held as the experimenter inserted the injector. The injector projected 1 mm beyond the guide cannula and 0.5 µl solution was infused in the right CA1 region over a 60 sec period. The injector was left in place for an additional 60 sec to allow diffusion of the solution and to reduce the possibility of reflux.

Elevated plus-maze test of anxiety
The method is mainly the same that was reported previously (16). The elevated plusmaze (EPM) comprised 2 open arms (50×10 cm) and 2 enclosed arms (50×10×40 cm) that extended from a common central platform (10×10 cm). An apparatus, constructed from wood, was elevated 50 cm above floor level. Testing was conducted in a quiet room between 9.00 a.m. and 13.00 p.m. Within 5 days following surgery, rats were brought into the behavioral testing room and left undisturbed for at least 1 hr prior to testing. The rats were individually placed in the center of the maze facing an open arm and allowed 5 min of free exploration. All sessions were videotaped. After each test, the floor was cleaned with distillated water. Animals were tested 5 min after microinjection of either AM 251, capsaicin or AMG 9810 and 30 min after microinjection of URB 597 (17). Thirty min is necessary to allow URB 597 to increase anandamide adequately in the CA1 region.

Experiment 2
The animals received intra-CA1 injections of vehicle or one of the three doses of AM 251 [0.01, 0.1 and 1 µg/rat, doses based on Roohbakhsh et al (17)]. Five min after the injections, animals were submitted to the EPM test (Figure 1, A3, B3, C3).

Experiment 3
The animals received intra-CA1 injections of vehicle or one of the three doses of capsaicin [0.003, 0.03 and 0.3 µg/rat, doses based on Terzian et al (20)]. Five min after the injections, animals were submitted to the EPM test (Figure 2, D1, E1, F1).

Experiment 4
The animals received intra-CA1 injections of vehicle or one of the three doses of AMG 9810 [0.003, 0.03 and 0.3 µg/rat, doses based on Pitcher et al (21)]. 5 min after the injections, animals were submitted to the EPM test. (Figure 2, D2, E2, F2).

Experiment 5
The animals received intra-CA1 injections of vehicle or one of the three doses of URB 597 [0.01, 0.1 and 1 µg/rat]. After 25 min, the rats received an injection of AMG 9810 [0.003 µg/rat]. Five min after the second injections, the animals were exposed to the EPM test. (Figure 1, A2, B2, C2).

Verification of cannula placements
After completion of the experimental sessions, 0.5 µl of methylene blue was injected in the CA1 region of the rats. Ten min after the injections, the animals were decapitated and their brains were removed, blocked and cut coronally through cannula placements. Data from the rats with injection sites located outside the dorsal hippocampus region were not used in the analysis.

Statistical analysis
One-way ANOVA was used for the comparison between the effects of different doses of drugs with vehicles. Two-way ANOVA was used for evaluation of interactions between drugs. Following a significant F-value, post-hoc analysis (Tukeytest) was performed for assessing specific group comparisons. Differences with P< 0.05 between experimental groups at each point were considered statistically significant.

Discussion
The main results of the present study demonstrated that both indirect activation of canabiniod receptors (by URB 597) and inhibition of TRPV1 receptors (by AMG 9810 ) in the CA1 region produced anxiolyticlike effects in the EPM while activation of TRPV1 receptors (by capsaicin) in this area induced anxiogenic-like effects. In addition, AMG 9810 did not change significantly the anxiolytic-like effects of URB597.
The current results showed that URB 597 produced anxiolytic-like effects in the CA1 region. In accordance with this finding, a number of previous studies have shown that inhibition of fatty acid amide hydrolase, the enzyme responsible for degradation of anandamide, induces anxiolytic-like effects in different animal models of anxiety. These findings have been reported following both peripheral (5,22) and intra-prefrontal cortex (19) administration of URB 597. Kathuria et al (5), for the first time, showed that URB 597 had anxiolytic-like effects in mice without a typical spectrum of cannabinoid responses such as catalepsy, hypothermia or hyperphagia. Meanwhile, it has been reported that URB 597 has antidepressant-like effects (23) and despite common cannabinoids and anti-anxiety drugs increases wakefulness (24) and it does not have reward-related behaviors (25). Adding all these findings up, we may propose that this new class of drugs would be a good candidate for making a new generation of anti-anxiety drugs. However, injection of URB 597 in the ventral hippocampus has been documented with an anxiogenic-like response (17). This controversial effect has also been reported by Rubino et al (19) at a high dose of URB 597. These contradictory results may represent the importance of injection site and/or the doses of FAAH inhibitors that have been employed in different studies.
In the present study, injection of AM 251 into the CA1 did not produce any significant effect. In line with this finding, Moreira et al (26) found that intra-dorsal periaqueductal grey administration of AM 251 did not modify animal behaviors in the elevated plusmaze. The same results have also been reported following intra-ventral hippocampus and peripheral administration of AM 251 (17,27). However, there are reports that have documented either an anxiogenic-like (28,29) or an anxiolytic-like effect (30,31) following administration of cannabinoid receptor antagonists. The above controversial results could be due to different methodological factors, sites of injections and different experimental conditions.
Our data showed that capsaicin, the active compound of hot chili, produced significant anxiogenic-like effects only at low and medium doses but not at the highest dose. A few numbers of the studies have evaluated the effect of TRPV1 agonists and antagonists on anxiety-like behaviors. Kasckow et al (10) showed that olvanil, a TRPV1 receptor agonist, induced anxiogenic-like effects in rats in the EPM test of anxiety. Conversely, capsazepine (a TRPV1 receptor antagonist) had anxiolytic-like effects in the same conditions. Rubino et al (19) also reported that infusion of capsaicin into the prefrontal cortex increased anxiety-like behaviors of the rats submitted to the EPM. In this study, capsaicin at the dose of 0.3 μg/rat (the highest dose) failed to show any significant change in animal behavior. Quick desensitization of TRPV1 receptors may be a possible explanation for this finding. Desensitization of TRPV1 receptors following administration of a high dose of capsaicin has been reported to be the reason for its analgesic effect after the initial hyperalgesia (32). This finding may suggest that TRPV1 receptors possibly have a modulatory role in the control of anxious states in the CNS.
We also showed that injection of TRPV1 receptor antagonist (AMG 9810) induced anxiolytic-like responses. In agreement with the results of this study, the anxiolytic-like effect of capsazepine following intra-ventral hippocampus (33), intra-dlPAG (20) and intra-prefrontal cortex (19) of the rats has also been reported previously. Furthermore, TRPV1 knockout mice have shown reduced anxiety-like behavior and impaired fear conditioning in the light-dark and EPM tests of anxiety (34). It is noteworthy that the TRPV1 receptor antagonist that we used in this study has a better profile of receptor selectivity and greater antagonistic potency in comparison with capsazepine (35). This may give us a better deduction about the net effect of TRPV1 antagonists in the control of anxiety-like behaviors. The results also showed that AMG 9810 at the low dose of 0.003 μg/rat increased exploratory activity, as a possible confounding factor, of the rats. However, this increase did not happen at higher doses that decreased anxiety and the reason of this observation remains to be answered and we have no explanation for it.
It has been documented that anandamide is an endogenous ligand for both CB1 and TRPV1 receptors. It means that endocannabinoids by two different mechanisms may show their physiological roles in the body. This has been the topic of a number of studies. For example, the involvements of TRPV1 receptors in the effects of anandamide on blood pressure (36) and movement (37) have been reported. To test the hypothesis that TRPV1 receptors may be involved in those effects of anandamide in the modulation of anxiety-like behaviors in the CA1 region, we injected an ineffective dose of AMG 9810 on anxiety-like behaviors with three different doses of URB 597 as an indirect agonist of endocannabinoid system. Statistical analysis did not show any significant difference in animal behaviors in comparison with the rats that received URB 597 alone. In a recent study, Rubino et al (19) showed that pharmacological blockade of TRPV1 receptors by capsazepine reversed the anxiogenic effect of URB 597 in the prefrontal cortex of the rats. A possible explanation for this finding is that TRPV1 receptors are activated mainly at high concentrations of anandamide that induces anxiogenic responses. This hypothesis can be further supported by the results of Rubino et al (19) that showed the anxiolytic-like effect of methanandamide at low but not high doses is mediated by CB1 receptors. The bimodal action of anandamide on anxiety-like responses has been reported in a separate study (38). With this view, a new generation of anxiolytic drugs that have both FAAH inhibitory and TRPV1 antagonistic activity was developed recently (39). More experiments are necessary to elucidate the exact mechanisms underlying the effects of endocannabinoids in the modulation of fear or anxiety-like behaviors.

Conclusion
FAAH enzyme inhibitors are a new class of drugs that may produce anxiolytic-like effects. These drugs increase wakefulness despite conventional anti-anxiety drugs and have a possible antidepressant effect that will increase their efficacy in clinical practice. Moreover, TRPV1 receptors may also have an important role in the regulation of anxietylike behaviors and our knowledge about their interaction with endocannabinoids may help better production of more efficient antianxiety drugs.